Antimycin A Stimulation of Rate-limiting Steps ...

[Pages:6]Plant Physiol. (1972) 49, 411-416

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Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis

in Isolated Spinach Chloroplastst

Received for publication September 13, 1971

BERNICE SCHACTER2 I AND JAMES A. BASSHAM

Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley,

California 94720

ABSTRACT

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of C02 in the chloroplasts, nor does it stimulate C02 fixation by increasing the level of the carboxylation substrate, ribulose-1, 5-diphosphate. Rather, it appears that antimycin A increases C02 fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1, 5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.

Antimycin A has been reported to stimulate the rate of photosynthesis of isolated spinach chloroplasts, as measured by the rate of bicarbonate uptake (11, 12, 15, 26). Identification of the site of action of this stimulation and determination of the mechanism of stimulation could aid in the general understanding of the control of the photosynthetic metabolism.

The action of antimycin A as an inhibitor and uncoupler of photoelectron transport and phosphorylation in chloroplast fragments has been previously reported (2, 3, 14, 16, 17, 28, 29). Several observations have led to proposals that antimycin A stimulates the rate of CO2 fixation in intact chloroplasts by stimulating the transport or activation of CO2 or bicarbonate for the carboxylation step (12, 26).

1 This work was supported in part by the United States Atomic Energy Commission.

2 Charles F. Kettering Postdoctoral Fellow, 1970-1971. 'Present address: Department of Pharmacology, University of Miami, Miami, Fla. 33136

The effects of antimycin A addition on the levels of labeled metabolites formed during photosynthesis by isolated, whole spinach chloroplasts are examined in this report. Also, the effects of antimycin A on the transport of metabolites from the chloroplasts were followed to determine if any changes in transport could be related to the stimulatory effects of antimycin A.

MATERIALS AND METHODS

Chloroplasts were isolated from spinach (Spinacia oleracea) leaves, and the rate of '4CO2 fixation was determined as previously described, with the exception that unless noted, isoascorbate was omitted from all solutions (18). Analysis of the "C and 'P-labeled products of photosynthesis by two-dimensional chromatography and radioautography was carried out as described earlier (23). Estimation of the distribution of metabolites between the chloroplasts and the surrounding medium was also carried out as previously described (8). The "4C radioactivity found at the origin was taken as a measure of the synthesis of "C-labeled starch and other polysaccharides. With whole cells, following photosynthesis with "CO2, acid hydrolysis of this insoluble material at the origin gives glucose, amino acid, and nucleic acid components (20, 21). Spinach chloroplasts, isolated and allowed to photosynthesize with "CO2 as described previously (12), produce essentially only polysaccharides as insoluble end products, since such chloroplasts form only negligible amounts of amino acids.

Chlorophyll was determined by the method of Arnon (1). Antimycin A was obtained from Sigma Chemical Co. and dissolved in absolute ethanol. The total concentration of ethanol was kept at 1 % or less, and, unless indicated, all controls contained 1 % ethanol.

RESULTS

In the earlier reports of the antimycin A stimulation of CO2 fixation with chloroplasts, the control rates were generally low-10 to 35 ,wmoles CO2 fixed/mg chlhr (11. 12, 26). An early observation in the present study was that chloroplasts with CO2 fixation rates of 130 ,umoles/mg chl-hr were stimu-

lated by 0.5 [kM antimycin A to C02 fixation rates of 180

,umoles/mg chl-hr. While the control rates and the magnitude of the antimycin A stimulation varied with the spinach used,

the presence of antimycin A (0.5-5.0 [LM) in the incubation

mixture from the start of the experiment dependably gave large stimulations over the control rate. A typical result is given in Figure 1. In the case where antimycin A was added at the beginning of the experiment the CO2 fixation rate (118 ,umoles/mg chl 'hr) during the first 5 min was stimulated 79% over the control rate. When antimycin A was added after 5-

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SCHACTER AND BASSHAM

Plant Physiol. Vol. 49, 1972

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FIG. 1. Rates of CO2 fixation by isolated spinach chloroplasts

with added antimycin A. (broken line): Control with 10 gl

ethanol added (rate = 66,moles C02/mg ch* l hr during

min); (solid line): control with 10 Al of ethanol added at min

,umoles (rate = 53

C02/mg chl-hr during 7- to 12-min interval);

A (broken line): antimycin A added at the beginning of experiment

(rate

,umoles 118

CO2/mg chl-hr during first 5 min); A (solid

line): antimycin added at 5 min (rate 106 /moles CO2/mg chl-hr

during 7- to 12-min interval).

min photosynthesis, the subsequent rate (106,umoles/mg

chl-hr) was 100% higher than the control rate (53,umoles/mg

chl -hr).

The experiment which gave the data shown in Figure 1 was

performed with no preincubation and an argon gas phase. Ad-

dition of antimycin A to chloroplasts after the usual light, air,

preincubation and 2 to 5 min photosynthesis in the presence

of bicarbonate sometimes gave little or no stimulation. Exclu-

air sion of

be seems to

required to obtain the stimulation re-

producibly. Moreover, the inclusion of 10 mm isoascorbate,

usually a standard component of the incubation mixture for

chloroplasts (18), also prevented the stimulatory response to

antimycin A, even if the antibiotic were initially present. Un-

less noted, therefore, isoascorbate was routinely omitted from

the reaction mixture, and an argon atmosphere was provided.

The effects of the addition during photosynthesis of anti-

mycin A on the incorporation of"4C into several intermediates

of the Calvin cycle are shown in Figures 2 to 8. The increase

in 3-PGA and RuDP, and the drop in SDP and in FDP, oc-

curred consistently upon the addition of antimycin A, but the

increase in starch and decrease in F6P occurred only if the

addition caused an increase in the rate of fixation.'

If antimycin A were simply increasing the effective concen-

tration of the CO2 substrate of the RuDP carboxylase, the

level of RuDP would drop upon stimulation. This did not oc-

cur (Fig. 4). In order to test whether or not a higher level of

chloroplasts CO2 inside the

could produce effects similar to

those caused by antimycin A addition, the incorporation

14C into the Calvin cycle intermediates accompanying a switch

from low to high bicarbonate concentrations were studied. The

changes in RuDP and SDP levels resulting from such

4Abbreviations: DHAP: dihydroxyacetone phosphate; FDP:

fructose- 6-diphosphate; FDPase: fructose- 6-diphosphatase

(E(C. 3.1.3.11); F6P: fructose-6-phosphate; PGA: 3-phosphoglyc-

erate; RuDP: ribulose-1 5-diphosphate; RuDP carboxylase: ribulose diphosphate carboxylase (E.C. 4.1.1.39); SDP: sedoheptulose-1,7-

diphosphate.

creased bicarbonate (Figs. 9 and 10) are completely different

from the changes due to antimycin A addition (Figs. 4 and 5).

The increase in SDP with increased bicarbonate concentration

was typical of the changes in all intermediates examined

(starch, PGA, DHAP, FDP, F6P, etc.) except for RuDP,

which clearly, and as predicted, dropped upon increasing the

bicarbonate concentration.

As previously reported (12, 26), antimycin A stimulated the

rate of CO2 fixation at both limiting and saturating bicarbon-

ate concentrations. The metabolic consequences of added anti-

mycin A were also similar at both bicarbonate concentrations,

as shown by the levels of SDP and RuDP in Figures 11 and

12.

(PPi,Since it had been demonstrated that regulatory factors

Mge, and a factor associated with spinach alkaline

FDPase) can have marked effects on the transport or release

of metabolites from the chloroplasts into the surrounding me-

dium (6), it was of interest to determine if antimycin A might

be exerting its stimulatory effects by changing the pattern of

transport of metabolites from the chloroplasts. An examina-

tion of the distribution between chloroplasts and the surround-

ing medium of the Calvin cycle intermediates labeled during

photosynthesis showed no significant changes in movement of

metabolites into the medium in response to antimycin A.

Because antimycin A has frequently been demonstrated to

uncouple both cyclic and noncyclic photophosphorylation in

chloroplast fragments (14), we examined the effect of anti-

mycin A on the levels of ATP in intact chloroplasts before

and during photosynthesis by following the incorporation of

'P, into ATP both in the presence and absence of bicarbon-

ate. The data presented in Figure 13 were obtained by incu-

bating chloroplasts in the presence and absence of 0.5 /M

antimycin A, first during an air, light preincubation, and then

after the addition of bicarbonate. There was no significant

change in the accumulation of 'P1 into ATP in the absence

of bicarbonate. Upon addition of bicarbonate, a slightly greater

decrease in the labeled pool of ATP was seen in the chloro-

plasts incubated with antimycin A. Since the rate of CO2 fixa-

,umoles tion was increased in this instance by antimycin A from 74 to

141

fixed/mg chl hr, the actual rate of synthesis of

ATP must be correspondingly greater in the chloroplasts in-

cubated with antimycin A than in the control chloroplasts.

Thus the decreased level of ATP might be due only to the

increased demand for ATP compared to its rate of synthesis.

This may also account for the drop in labeled ATP observed

upon the addition of antimycin A to photosynthesizing chloro-

plasts (Fig. 14).

It was also found that the stimulation of CO2 fixation by

antimycin A is somewhat sensitive to the concentration of

added Mg2_ (Fig. 15). Stimulationof CO2 fixation by 0.5 /IM

antimycin A decreased appreciably with increasing Mg2+ con-

/tMcentration above about 0.3mM Mg2+. The stimulation by 5 antimycin A was relatively insensitive to added Mg2+. Un-

like the inhibitory fraction obtained from spinach juice (6). antimycin A is not synergistically inhibitory with Mg2+.

DISCUSSION

That antimycin A might increase the chloroplasts' efficiency for utilization of bicarbonate had been suggested primarily by the following observations: (a) the increase in both the absolute and relative amount of synthesis of PGA in the presence

of antimycin A (11, 26); (b) the decrease in the Km(HCO3,)

of the chloroplasts in the presence of antimycin A (12, 26); (c) the apparent insensitivity of the RuDP carboxylase to antimycin A when assayed in osmotically lysed chloroplasts

(26): (d) the fact that the Km(HCO,,) of the carboxylase is

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Plant Physiol. Vol. 49, 1972

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